Methylmercury (MeHg) is a persistent environmental neurotoxin that poses a health risk to humans due to its accumulation in dietary fish. Epidemiological and laboratory studies have established that the developing nervous system is exceptionally sensitive to MeHg toxicity. Our overall goal is to elucidate the fundamental mechanisms of neural development that are targeted by MeHg toxicity. The extent to which MeHg selectively alters neurogenesis versus cell fates, neuronal migration and/or neuron morphology remains unclear. It is well understood, however, that these neural developmental events are temporally regulated over the course of embryogenesis. Our hypothesis is that MeHg will show a preferentially higher activity toward disruption of one of four neural developmental events: neuroblast specification, neuron sibling cell fates, neuronal/glial migration or neuron morphogenesis. We therefore predict that susceptibility of the embryonic nervous system to MeHg toxicity will vary with the developmental timing of MeHg exposure. We will test this hypothesis with the Drosophila embryo model. Our study is made feasible by our breakthrough innovation of an embryo permeabilization solvent (EPS) that overcomes the longstanding technical barrier of permeating of the fruit fly eggshell while maintaining embryo viability. EPS treatment enables delivery of defined doses of small molecules to embryos cultured in vitro. By applying MeHg to the Drosophila embryo at discrete developmental time points and monitoring phenotypes in well characterized neural lineages we expect to elucidate whether early (neurogenesis and cell fate specification) or late (neuron migration and morphogenesis) development events are the most susceptible to MeHg toxicity. As EPS is a new application we will first take steps to calibrate levels of embryo permeability and perform MeHg dosimetry with treated embryos. We will then apply this methodology to available transgenic fly strains with lineage-specific reporter genes to determine the most vulnerable neural developmental mechanisms. Since many of the signaling pathways underlying neural development in the fly embryo are highly conserved, we expect these results will drive the rationale for more focused molecular studies on MeHg targets in higher organisms and in humans. Our novel embryo permeabilization innovation is an enabling step in executing accurate doses to the fly embryo. Therefore, we also view this study as essential for establishing the utility of this experimental approach for the broader toxicological research community. PUBLIC HEALTH RELEVANCE: Methylmercury is an environmental neurotoxin that disrupts development of the nervous system in the fetus and young children. This proposal seeks to identify the most methylmercury-susceptible mechanisms of neural development during the course of embryogenesis. Knowledge of these mechanisms will advance our ability to determine and advise people who are most at risk when exposed to methylmercury.